TP CH PHT TRIN KH&CN, TP 12, S 10 - 2009

CONTRIBUTION TO THE STUDY ON CHEMICAL CONSTITUENTS OF

HYDROCOTYLE VULGARIS (L.), APIACEAE
Ton Nu Lien Huong(1), Nguyen Kim Phi Phung(2), Nguyen Ngoc Suong(2)
(1) Can Tho University
(2)University of Science, VNU-HCM
(Manuscript Received on January 08th, 2009, Manuscript Revised September 04 th, 2009)

ABSTRACT: The essential oil of Hydrocotyle vulgaris was analyzed by GC-MS then
tested citotoxicity on the cancer cells. The result showed that the essential oil of Hydrocotyle
vulgaris had weaker bioactities than the one of Hydrocotyle bonariensis, and in two species
had the same main compounds. In addition, from the ethyl acetate extract, quercetin 3-Ogalactopyranoside was isolated and identified by the spectrum data 1D, 2D-NMR and MS.
Key works: Hydrocotyle vulgaris, quercetin 3-O-galactopyranoside, bioactivities on RD,
Hep-G2 and LU cancer cells.
OH
3'

HO

1'

OH

4'

2'

OH

O
2''

1''

OH

5''

HO

OH
4''

OH

(1)
Hydrocotyle vulgaris

Hydrocotyle bonariensis

1. INTRODUCTION
Hydrocotyle vulgaris (marsh pennywort, water pennywort, Rau ma la sen) is a new plant
growing in the Mekongdelta and used with H. asiatica, H. sibthorpioides as a jumble
vegetable. Hydrocotyle and Centella species (Apiaceae) produce characteristic essential oils
throughout the whole plant. It is known that H. sibthorpioides Lam and H. maritima Honda
have hemostatic and antitumor activities [1]. Besides, H. vulgaris has the botanical aspect
similar to the one of the species H. bonariensis.
In the continuation of the study on Hydrocotyle genus, now we report the chemical
constituents of H. vulgaris and their bioactivities in vitro on RD and Hep-G2, LU cancer cells.
These compounds of essential oil and quercetin 3-O-galactopyranoside were identified by the
spectrum data NMR, MS or GC-MS.
2. RESULT AND DISCUSSION
Compound (1) was isolated from the ethyl acetate extract and presented as yellow crystals.
Its TLC, showed indigo-blue spot when the layer was sprayed by solution FeCl3 5% in

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Science & Technology Development, Vol 12, No.10- 2009

methanol, so perhaps (1) was a flavonoid compound. The EI-MS gave a peak with m/z = 464.6
[M]+. IR, KBr, cm-1: 3399 (O-H), 2923 (C-H), 1688 (C=O flavone), 1606 (C=C). 1H-NMR
(500 MHz, DMSO) ppm: 3.2 5.0 (protons of sugar), 6.2 (1H, d, J = 1.5 Hz, =CH); 6.9 (1H,
d, J = 8.5 Hz, -CH=), 7.7 (1H, =CH). 13C-NMR spectral data showed 21 carbons. The
chemical shiffs ppm of the carbons were: 177.2 (-C=O), 164.1 (=C-OH), 161.2 (=C-OH),
148.4, 144.8, 133.4, 121.9, 115.1, 98.6, 93.4. These signals manifested that (1) was a flavonol.
In additon, the present of sugar was showed by the chemical shiffs of the (H-1 H-4)
protons at (3.4 5.2) ppm and of the (C-1C-6) in turn carbons at ppm: 101.8 (carbon
anomer), 71.2 , 73.1, 75.8 and 67.9 (CH-OH), 60.1 (CH2-OH). The sugar was suggested as
galactopyranose. The spectral data were showed on the table 1. The HMBC spectrum showed
long-range coupling between H-1 with C-3 so the sugar linked to the flavonol at its C-3. Base
on the 1D and 2D-NMR, as well as comparison with the authentic sample, in one previously
reported of Hydrocotyle sibthorpioides [4], compound (1) was identified as quercetin 3-Ogalactopyranoside.
Table 1. 13C NMR ppm values in DMSOd6 solutions
Assigned
position
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-1
C-2
C-3
C-4
C-5
C-6
C-1
C-2
C-3
C-4
C-5
C-6

Compounds
(1)
156.2
133.4
177.4
161.2
98.6
164.1
93.4
156.2
103.9
121.9
115.1
144.8
148.4
115.9
121.0
101.8
71.2
73.1
67.9
75.8
60.1

Quercetin
3-galactoside
156.3
133.4
177.4
161.1
98.6
164.0
93.5
156.3
103.9
121.9
115.1
144.7
148.4
115.9
121.0
101.9
71.2
73.1
67.9
75.7
60.1

Hydrcotyle vulgaris was divided

into stem, leave and flower and each
part was subjected to the steam
distillation. Each essential oil was
analyzed by GC-MS and the results
were presented in table 2. The major
components of essential oil of the stem
and the flower are the same with the
presence of some sesquiterpenes such
as:
santalene,
-farnesene,
sesquiphellandrene, -bisabolene. On
the contrary, the essential oil of leave
contained alcohol and aldehyde such as
3-hexen-1-ol and 2-hexenal with the
great
amount, like the one of H.
bonariensis.
The essential oil was evaluated the
antimicrobial activity on E. coli, P.
aeruginosa, S. aureus, F. oxysporum
(table 3) and was also tested the
cytotoxicity on the RD, Hep-G2, LU
cancer cells by the Likhiwitayawuid
assay (table 4). The essential oil of H.
vulgaris had weaker bioactivities than
the one of H. bonariensis.

Table 2. The essential oil of H. vulgaris

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TP CH PHT TRIN KH&CN, TP 12, S 10 - 2009

Peak
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

Rt
Compounds
3.38
Hexenal
(2E)-Hexenal
3-Hexen-1-ol
Santalene
-Farnesene
-Cubebene
-Muurolene
-Bisabolene
-Sesquiphellandrene
Nerolidol
Caryophyllene oxid
Ledol
Z--Bisabolene epoxide
Globulol
epi-Globulol
5,5-Dimethyl-4-(3-methyl-1,3butadienyl)-1-oxaspiro[2.5]octane

4.23
4.36
10.61
10.78
10.91
10.96
11.05
11.13
11.28
11.54
11.69
11.71
11.73
11.82
11.89

Leaves
(%)
6.59

Stem
(%)
-

Flower
(%)
-

18.53
36.01
10.07
6.84
-

29.81
21.71
7.96
14.68
20.68
5.16
-

29.51
21.42
8.25
14.46
19.56
1.18
0.77
3.71
0.74
0.41

7.90
7.32
1.19
5.55

Table 3. Result of the antimicrobial assay of the essential oil.

Sample
Ess. oil of H. bon..
Ess. oil of H. vul..

Bacteria Gram (-)

E.
P.
coli
aeruginosa
100
200
(-)
(-)

MIC (g/ml)
Bacteria Gram (+)
Fungus
B. subS.
Asp. F. oxytillis
aureus niger sporum
(-)
100
(-)
100
(-)
(-)
(-)
(-)

Yeast
S.cereC.
visiae
albicans
(-)
(-)
(-)
(-)

Table 4. Result of the cytotoxicity test on cancer cells of the essential oil.
No

Sample

Cell survival (%)

Hep-G2

IC50 (g/ml)

LU

RD

Hep-G2

RD

DMSO

100.0 0.00

100.0 0.00

100.0 0.00

Ellipithine

3.00 0.01

1.50 0.00

1.70 0.00

0.19

0.10

Essential oil of H. bon.

50.0 0.50

84.59 1.00

6.87 0.60

19.93

16.10

Essential oil of H. vul.

100 0.00

100 0.00

88.9 0.55

> 20

3. EXPERIMETAL
3.1. General
1
H- and 13C-NMR were recorded on Bruker Avance 500 MHz and 125 MHz, respectively,
in MEOD. LC-MS spectra were carried out on Agilent-MSD-Trap-SL with the column of
Adserbphere UHF C18. All spectrums were recorded in the Institute of Chemistry, Vietnamese
Academy of Science and Technology, Cau Giay Dist., Ha Noi.

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GC-MS spectra were obtained under the following conditions, GC column: 30 m x 250
m, Agilent 6890N; MD Agilent 5973 inert; temp. 60o 280oC at 10o/min. after the first 3
minutes and 30o/min. after 100o C, injector temp. 250oC, He 0.9 ml/min., p = 6.9 psi.
The essential oil was evaluated the antimicrobial activities on E. coli, P. aeruginosa, S.
aureus, F. oxysporum and cytotoxic activities on the RD, Hep-G2, LU by the Likhiwitayawuid
assay.
3.2. Plant material
The leaves of Hydrocotyle vulgaris L. were collected in Tien Giang province in December
2007. A voucher specimen was determined by Mr. Phan uc Binh, Assistant editor of Journal
Medicine and HealthHo Chi Minh City and was deposited in Department of Sciences, Can
Tho University.
3.3. Extraction and isolation
Dried and powdered whole plant of Hydrocotyle vulgaris (1800 g) was macerated in
ethanol 95% at room temperature to give methanol extract (150 g). This crude extract was
separated into petroleum ether, chloroform, ethyl acetate and methanol extracts, respectively,
by the technique of silica gel solid phase extraction. The ethyl acetate extract (1.20 g) was
subjected to column chromatography, eluted with different solvents, yielded 16 fractions (E1.1
E1.16). From the fraction E1.12, the quercetin 3-O-galactoside was isolated (1, 140 mg).
3.4. Quercetin 3-O-galactoside
Yellow crystals from MeOH; mp.:230 232oC; EI-MS, m/z = 464.6 [M]+ (C21H20O12 , M=
KBr
464); IR, max
cm-1: 3399 (O-H), 2923 (C-H), 1688 (C=O flavone), 1606 (C=C); 1H-NMR
(500 MHz, DMSO) ppm: 7.7 (1H, d, J = 2.7 Hz, H2), 7.6 (1H, dd, J = 2.7 and 8.1 Hz, H6),
6.9 (1H, d, J = 8.5 Hz, H5), 6.4 (1H, d, J = 1.5 Hz, H8), 6.2 (1H, d, J = 1.5 Hz, H6), 3.2 5.0
(protons of sugar); 13C-NMR (125 MHz, DMSO) ppm: 177.2 (C-4), 164.1 (C-7), 161.2 (C5), 156.2 (C-2), 156.2 (C-9), 144.7 (C-3),148.4 (C-4), 121.9 (C-1), 121.0 (C-6), 115.1(C5), 103.9 (C-10), 101.8 (C-1), 98.6 (C-6), 93.4 (C-8), 75.8 (C-5), 73.1(C-3), 71.2 (C-2),
67.9 (C-4) and 60.2 (C-6).
3.5. Biotest
Whole raw plant of H. vulgaris was divided into stem, leave and flower and each part was
subjected to the steam distillation. The essential oils are faint aroma light yellow oil, with
small content (0,057 %), ( d 30 ): 0,737, ( n D30 ): 1,571. Each essential oil was studied by GCMS. Because of the lack of sample, the essential oil of different parts of the plant were mixed
together and then was evaluated the antimicrobial activities and cytotoxic activities on the RD,
Hep-G2, LU cancer cells.

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TP CH PHT TRIN KH&CN, TP 12, S 10 - 2009

GP PHN TM HIU THNH PHN HA HC CY RAU M L SEN

HYDROCOTYLE VULGARIS (L.), H NG (APIACEAE)
Tn N Lin Hng(1), Nguyn Kim Phi Phng(2), Nguyn Ngc Sng(2)
(2) Trng i Hc Cn Th
(2)Trng i Hc Khoa Hc T Nhin, HQG -HCM
TM TT: Cy Rau m l sen, Hydrocotyle vulgaris, l loi mi pht hin vng ng
bng sng Cu Long, c s dng xen ln vi cc loi rau m khc lm rau n. Cc cy Rau
m l sen c hnh dng tng t nhau rt d nhm ln khi thu hi, v cc cy Hydrocotyle
vulgaris v Hydrocotyle bonariensis c hnh dng rt ging nhau ch khc hoa. Hydrocotyle
bonariensis cha c kho st trn th gii, cn Hydrocotyle vulgaris cha c kho st
Vit Nam.
Nhm tip tc cc nghin cu trn chi Hydrocotyle, tip theo phn bo co v
Hydrocotyle bonariensis, trong bi bo ny chng ti trnh by v thnh phn ha hc v hot
tnh khng cc vi sinh vt, c tnh khng t bo ung th RD, Hep-G2, LU in vitro ca cy
Hydrocotyle vulgaris. Cc cht c nh danh t cc d liu ph NMR, MS hoc GC-MS.
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HCM City, pp. 631 632, (2004).
[2].Vo Van Chi, Tran Hop, Vietnamese useful plants, Vol 1, Educational Publishing
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[3].Ton Nu Lien Huong, Nguyen Ba Vi, Nguyen Kim Phi Phung, Nguyen Ngoc Suong,
Chemical constituents of volatile oil and organic extract from Hydrocotyle bonariensis
(Apiaceae), Poster No 52, ASIA-LINK medicinal conference, (2006).
[4].Myata, S. Anticancer crude drugs and their prescrition sp. 185-186,
Kagakushoin,Tokyo (in Japanese) (1981)
[5].Nakaoki, T. and Morita, N., Yakugaku Zasshi, 80, 1474, (1960)
[6].Shigematsu Nobuharu, Kouno Isao, Kawano Nobusuke, Quercetin 3-galactoside,
Quercetin 3-(6-caffeoylgalactoside) from Hydrocotyle sibthorpioides, Phytochemistry
21(8), 2156-2158, (1982).

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